The structural basis of Holliday junction resolution by T7 endonuclease I

Abstract

The four-way (Holliday) DNA junction is the central intermediate in homologous recombination, a ubiquitous process that is important in DNA repair and generation of genetic diversity1. The penultimate stage of recombination requires resolution of the DNA junction into nicked-duplex species by the action of a junction-resolving enzyme, examples of which have been identified in a wide variety of organisms2. These enzymes are nucleases that are highly selective for the structure of branched DNA. The mechanism of this selectivity has, however, been unclear in the absence of structural data. Here we present the crystal structure of the junction-resolving enzyme phage T7 endonuclease I in complex with a synthetic four-way DNA junction. Although the enzyme is structure-selective, significant induced fit occurs in the interaction, with changes in the structure of both the protein and the junction. The dimeric enzyme presents two binding channels that contact the backbones of the junction’s helical arms over seven nucleotides. These interactions effectively measure the relative orientations and positions of the arms of the junction, thereby ensuring that binding is selective for branched DNA that can achieve this geometry.

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Figure 1: Schematic representation of the Holliday junction in the crystal structure.
Figure 2: The structure of the complex and the active site.
Figure 3: Local distortion at the branch point.

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Acknowledgements

We thank D. Flot at the ESRF for his assistance with the X-ray data collection and V. Sergeant for technical support. This work was supported by funds from the Wellcome Trust, BBSRC and Cancer Research UK.

Author Contributions J.M.H. purified and crystallized the complex, collected data, and built and refined the structure. A.-C.D. designed DNA oligonucleotides for co-crystallization and performed the molecular biology and biochemical studies. S.B.C. collected data and refined the structure of the complex. D.M.J.L. and S.E.V.P. conceived and designed the study and also helped to analyse the data. All authors discussed the results and wrote the paper.

Atomic coordinates and structure factors have been deposited in the PDB database under the accession code 2PFJ.

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Correspondence to Simon E. V. Phillips.

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Reprints and permissions information is available at www.nature.com/reprints. The authors declare no competing financial interests.

Supplementary information

Supplementary Information

The file contains Supplementary Tables 1-3, Supplementary Figure 1 with Legend and legends to Supplementary Videos 1 and 2. (PDF 596 kb)

Supplementary Video 1

The file contains Supplementary Video 1 showing rotating view of the Endo I binding site, formed by an alignment of the major grooves of DNA helices within the complex. (MOV 13821 kb)

Supplementary Video 2

The file contains Supplementary Video 2 depicting the re-orientation of the protein domains on binding the 4-way DNA junction. (MOV 3470 kb)

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Hadden, J., Déclais, A., Carr, S. et al. The structural basis of Holliday junction resolution by T7 endonuclease I. Nature 449, 621–624 (2007). https://doi.org/10.1038/nature06158

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